The present invention relates to processes for establishing multi-tier platforms in rolling circle amplification so as to provide enhanced detection of product species with quantitative and kinetic advantages over previous rolling circle methods.
A means of amplifying circular target DNA molecules is of value because such amplified DNA is frequently used in subsequent methods including DNA sequencing, cloning, mapping, genotyping, generation of probes, and diagnostic identification.
Heretofore, several useful methods have been developed that permit sensitive diagnostic assays based on detection of nucleic acids. Most are designed around the amplification of selected targets and/or probes composed of DNA, including the polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), and amplification with Qxcex2 replicase (Birkenmeyer and Mushahwar, J. Virological Methods, 35:117-126 (1991); Landegren, Trends Genetics, 9:199-202 (1993)). Some of these methods suffer from relatively low precision in quantitative measurements, especially noticeable in multiplex assays (where more than one target is to be assayed simultaneously). These shortcomings have been largely overcome by rolling circle amplification (RCA) methods.
Previously, several methods have been employed to amplify circular DNA molecules such as plasmids or DNA from bacteriophage such as M13. One has been propagation of these molecules in suitable host strains of E. coli, followed by isolation of the DNA by well-established protocols (Sambrook, J., Fritsch, E. F., and Maniatis, T. Molecular Cloning, A Laboratory Manual, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). PCR has also been a frequently used method to amplify defined sequences in DNA targets such as plasmids and DNA from bacteriophage such as M13 (PCR Protocols, 1990, Ed. M. A. Innis, D. H. Gelfand, J. J. Sninsky, Academic Press, San Diego.) Some of these methods suffer from being laborious, expensive, time-consuming, inefficient, and lacking in sensitivity.
As an improvement on these methods, linear rolling circle amplification (LRCA) uses a primer annealed to the circular target DNA molecule and DNA polymerase is added. An improvement on LRCA is the use of exponential RCA (ERCA), with additional primers that anneal to LRCA product strand. Therefore, double stranded DNA can be produced, and exponential amplification can occur via strand displacement reactions referred to a HRCA (Lizardi, P. M. et al. Nature Genetics, (1998) 19. 225-231).
The multiple targets of multiplexed assays may, for example, be viruses or other microorganisms. Thus, the clinical condition of a virus-infected patient may depend heavily on viral load (for example, in HIV infections) and so a means for quantitatively determining such viral load is of especial value. In such multiplex assays, it is important that measurements of different targets, such as different viruses, or different strains of virus, be accurately determined and that the ratio of different targets be a true indicator of the ratio of the target sequences. For such purposes, multiplexed, exponential nucleic acid amplification methods have often been employed, but only multiplexed rolling circle amplification has been successful in meeting many of the goals of multiplexed assay systems [See: Lizardi, U.S. Pat. No. 5,854,033 the disclosure of which is hereby incorporated by reference in its entirety].
However, there are sources of error in such methods, such as where structural differences lead to different efficiencies, for example, different events are involved for different target sequences, or differences in the rates of product strand annealing may differ for different target sequences and lead to varying rates of competition with the aforementioned priming events, the effects of having multiple ligation effects occurring simultaneously for species of differing structure and stability (which events may be magnified by repetition of the ligation reactions), and the possibility that small differences in yield from one cycle of amplification to another may be magnified exponentially to result in undesirably large differences in the ratios of the final product. RCA methods have overcome errors due to signal yields, since amplification yields are proportional to the amount of target (i.e., detection efficiency is not dependent on the availability of ample amounts of target DNA so that even minute amounts of target can provide enormous signal sensitivity).
The earliest method for DNA amplification was the polymerase chain reaction (PCR) which operated only on linear segments of DNA and produced linear segments using specific primer sequences for the 5xe2x80x2- and 3xe2x80x2-ends of a segment of DNA whose amplification was desired. As an improvement on this method, linear rolling circle amplification (LRCA) uses a target DNA sequence that hybridizes to an open circle probe to form a complex that is then ligated to yield an amplification target circle and a primer sequence and DNA polymerase is added. The amplification target circle (ATC) forms a template on which new DNA is made, thereby extending the primer sequence as a continuous sequence of repeated sequences complementary to the ATC but generating only about several thousand copies per hour. An improvement on LRCA is use of exponential RCA (ERCA) with additional priming sequences that bind to the replicated ATC-complement sequences to provide new centers of amplification, thereby providing exponential kinetics and greatly increased amplification. Exponential rolling circle amplification (ERCA) employs a cascade of strand displacement reactions but is limited to use of the initial single stranded RCA product as a template for further DNA synthesis using individual single stranded primers that attach to said product but without additional rolling circle amplification.
All of these methods suffer from a lack of sensitivity, especially to rare genetic events, such as infrequent mutations, as well as to limits on multiplexing and the availability of flexible detection procedures.
The method of the present invention (referred to herein as Poly-Primed Rolling Circle Amplificationxe2x80x94PPRCA) avoids such disadvantages by employing a procedure that improves on the sensitivity of linear rolling circle amplification while retaining high specificity by employing true exponential amplification using additional stages, or platforms, of RCA (thereby affording greater sensitivity) while eliminating any reliance on a ligation step yet retaining the advantages of exponential RCA and the ability to amplify on a solid phase. The present invention has the advantages of being highly useful in new applications of rolling circle amplification, low cost, sensitivity to rare events, flexibility, especially in the use of detection reagents, and low risk of contamination.
The present invention relates to a process for the selective amplification of target DNA molecules as a means of detecting differences in the genotype of said DNA molecules, including changes, or mutations, as slight as a single nucleotide (a single nucleotide polymorphismxe2x80x94SNP) as well as providing a quantitative measure of the relative presence of such mutations in a given sample of DNA.
The present invention further provides a method for the selective detection of target DNA molecules by selective amplification thereof using secondary, tertiary, quaternary or higher order platforms especially designed to amplify selected sequences within the primary product of linear or exponential rolling circle amplification and amplifying said sequences along with specialized detector or reporter molecules that serve to enhance the ability to detect the amplification products.
In separate embodiments, the reporter molecules useful within the methods of the present invention include such molecules as biotin, digoxigenin, hapten and mass tags or any combination of these.
In other embodiments, the present invention employs selected nucleotides, or functionally equivalent structures, to provide linkages for detectors and reporter binding molecules of different kinds, such linkages utilizing different deoxynucleoside phosphates as well as a basic nucleotides and nucleosides selectively structured and configured so as to provide an advantage in detecting the resulting rolling circle products. Reporter molecules may also include enzymes, fluorophores and various conjugates.
A further aspect of the present invention relates to a process for the enhanced amplification of circular DNA targets. It improves on the sensitivity of linear rolling circle amplification with singly-primed template circular DNA molecules.